Lungs were fixed in formalin, paraffin-embedded, cut into 5-μm slices, and mounted on the slides. For fibrinogen immunohistochemical staining, deparaffinized slide-attached lung cross-sections were stained with polyclonal goat antiserum to mouse fibrin(ogen) 1:250 (Accurate Chemical & Scientific Corporation, YNGMFBGBio), followed by biotin-SP donkey anti-goat secondary antibody 1:600 (Jackson Immuno Research, 112-065-003). Stained lung cross-sections were subsequently scanned with a BX51 microscope equipped with virtual microscopy system dotSlide (Olympus, Japan). Image segmentation was performed in Ilastik (developed by the Ilastik team, with partial financial support by the Heidelberg Collaboratory for Image Processing, HHMI Janelia Farm Research Campus, and CellNetworks Excellence Cluster).
Dotslide
Manufactured by Olympus
Sourced in Japan, Belgium
The DotSlide is a digital slide scanning system designed for high-resolution imaging of microscope slides. It captures images of entire glass slides at high magnification, enabling comprehensive digital documentation and analysis of microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (3 mentions)
Rat anti mouse ly 6g mab, by BD (2 mentions)
Bx61vst, by Olympus (2 mentions)
Coreldraw x6, by Corel (2 mentions)
Methyl green, by Merck Group (1 mentions)
Histoplast, by Thermo Fisher Scientific (1 mentions)
Ab53519, by Abcam (1 mentions)
Tcs sp, by Leica (1 mentions)
Vs120 s1, by Olympus (1 mentions)
D2 40, by Abcam (1 mentions)
Anti pan keratin, by Merck Group (1 mentions)
Anti cd31, by Abcam (1 mentions)
Olyvia software, by Olympus (1 mentions)
M0851, by Agilent Technologies (1 mentions)
A0082, by Agilent Technologies (1 mentions)
Immpress hrp anti rabbit igg polymer detection kit, by Vector Laboratories (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Wheat germ agglutinin alexa fluor 488 conjugate wga, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
19 protocols using dotslide
1
Fibrinogen Immunohistochemistry in Lung Sections
2
Histological Analysis of Lung Inflammation
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Paraffin-embedded 4 µm tissue sections were stained with haematoxylin and eosin (H&E) and analyzed for inflammation and tissue damage as described [22] (link)–[25] (link). Briefly, all slides were coded and scored by a pathologist blinded for the experimental groups. Lung tissues were scored for the following parameters: interstitial inflammation, necrosis, endothelialitis, bronchitis, edema, pleuritis, presence of thrombi and percentage of lung surface with pneumonia. All parameters were rated separately from 0 (condition absent) to 4 (most severe condition). The total histopathological score was expressed as the sum of the scores of the individual parameters, with a maximum of 24. Granulocyte stainings, using fluorescein isothiocyanate-labeled rat-anti-mouse Ly-6G mAb (BD Pharmingen, San Diego, CA) were done as described previously [23] (link)–[25] (link). Slides were counterstained with methylgreen (Sigma-Aldrich, St. Louis, MO). The total tissue area of the Ly-6G-stained slides was scanned with a slide scanner (Olympus dotSlide, Tokyo, Japan) and the obtained scans were exported in TIFF format for digital image analysis. The digital images were analyzed with ImageJ (version 2006.02.01, National Institutes of Health, Bethesda, MD) and the immunopositive (Ly6G+) area was expressed as the percentage of the total lung surface area.
3
3D Reconstruction of Animal Nervous Systems
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Adult specimens were fixed (see Additional file 1 : Table S1 for species details), stained and analyzed as described in Beckers et al. [51 (link)]. Thus, the specimens were fixed overnight in Bouin’s fixative modified after Dubosque-Basil, dehydrated in an ethanol series and incubated in methylbenzoat and butanol. Afterwards the samples were pre-incubated in Histoplast (Thermo Scientific, Dreieich, Germany) and embedded in Paraplast (McCormick Scientific, Richmond, USA). 5 μm thick sections were made using a Reichert-Jung Autocut 2050 microtome (Leica, Wetzlar, Germany) and transferred to albumen-glycerin coated glass slides. Sections were stained with Carmaulaun, differentiated with sodium phosphotungstate (5%), washed in distilled water, stained in aniline blue orange G and subsequently embedded with Malinol (Waldeck, Münster, Germany). In Azan staining, the neuropil of the nervous system stains gray, the nuclei of cell somata stain red, the extracellular matrix stains blue and the musculature stains orange [51 (link)]. Each section was digitalized at 40× magnification using a slide scanner (Olympus dotslide (2.2 Olympus, Hamburg) and aligned using IMOD [52 (link)] and imodalign (http://www.q-terra.de/biowelt/3drekon/guides/imod_first_aid.pdf ). 3D reconstructions were performed with Fiji (1.45b) [53 (link)], trakem [54 (link)] and Amira (4.0).
4
Snail Expression in Lung Arteries
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Formalin-fixed and paraffin-embedded lungs were cut into 5-μm slices. Antigen retrieval was performed according to the standard protocol. To visualize expression of Snail, the slices were incubated with primary anti-Snail antibody (ab53519; Abcam) and secondary biotinylated donkey anti-goat antibodies (705-065-147; Jackson ImmunoResearch) concomitantly with ABC vector complex. For each slice, 10 randomly chosen nonobstructed arteries were photographed with a BX51 microscope (objective 20×) equipped with the virtual microscopy system dotSlide (Olympus, Japan) and the length of Snail-positive fragment(s) within the artery was manually measured and expressed as the percentage of the entire circuit of the particular artery. At the same time, the representative images of the investigated arteries were assessed for their patency (i.e., obstruction with blood clot or cancer cells) by a blinded investigator.
5
Histopathological Analysis of Lung Inflammation
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Paraffin-embedded 4 μm lung sections were stained with haematoxylin and eosin (H&E) and analyzed for inflammation and tissue damage, as described previously [19 (link)],[20 (link)]. All slides were scored by an experienced histopathologist blinded for experimental groups for the following parameters: bronchitis, interstitial inflammation, edema, endothelialitis, pleuritis and thrombus formation. All parameters were rated separately from 0 (condition absent) to 4 (most severe condition). The total histopathology score was expressed as the sum of the scores of the individual parameters, with a maximum of 24. Staining for granulocytes, using fluorescein isothiocyanate-labeled rat-anti-mouse Ly-6G mAb (BD Pharmingen, San Diego, CA) was performed as described previously [22 (link)],[23 (link)]. All slides were slightly counterstained with methylgreen. The total tissue area of Ly-6G stained slides was scanned with a slide scanner (Olympus dotSlide, Tokyo, Japan) and the obtained scans were exported in TIFF format for digital image analysis. The digital images were analyzed with ImageJ (version 2006.02.01, National Institutes of Health, Bethesda, MD) and the immunopositive (Ly6G+) area calculated from an average of 10 images per lung was expressed as the percentage of the total lung surface area.
6
Microscopic Imaging and Immunohistochemistry Protocol
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To image the NADPH-d stain in bright-field microscopy and to visualize immunohistochemical labeling, the sections were viewed with a VS120 S1 microscope [Olympus BX61VST with software dotSlide® (Olympus)]. For overviews in Figure 1A and in enlarged images of the immunohistochemical labeling, confocal optical sections were acquired with a Leica TCS SP confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) equipped with a Plan 10.0×/NA 0.40 and a Plan 63×/NA1.32 oil immersion objective. After stack acquisition and Z chromatic shift correction between color channels, RGB stacks, montages of RGB optical sections, and maximum-intensity projections were assembled into tables using ImageJ (1.39q Wayan Rasband, National Institutes of Health, USA) and Adobe Photoshop CS6 (Adobe Systems, San Jose, CA) software; figure images were arranged using CorelDRAW X6 (Corel Corporation, Ottawa, Ontario, Canada).
7
Hepatic Morphological Changes Assessment
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To provide an overview of changes in hepatic morphology, an experienced veterinary pathologist examined H&E-stained 5-μm liver sections. Alterations in hepatic lobular structure including the number of hepatic lobules per field of view and hepatic lobule area were measured on size-standardized, H&E-stained, liver sections using ImageJ software (National Institutes of Health, Bethesda, MD).13 Oil Red O staining14 was performed on frozen optimal cutting temperature–embedded 10-μm liver sections to visualize hepatic fat accumulation. After staining, a minimum of 10 individual hepatic lobules were photographed per pig (Leica Biosystems, Mount Waverly, Australia) and optical density measurements were performed using ImageJ software. Results are expressed as a percentage of Oil Red O staining per field of view obtained from a minimum of 10 fields of view per pig. Trichrome staining was performed on 4-μm formalin-fixed liver sections and the slides were scanned at ×20 magnification (Dotslide; Olympus Corporation, Tokyo, Japan). Changes in endothelial wall thickness were detected using ImageJ software on a minimum of 10 vessels per pig.
8
Quantifying Cholinergic Neurons in LSO
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For delineation of SOC nuclei and counting of cholinergic neurons, sections were imaged with a virtual slide microscope (VS120 S1, Olympus BX61VST, Olympus-Deutschland, Hamburg, Germany) at 10× magnification using the proprietary software dotSlide® (Olympus). All three colors of the secondary antibodies used for immunostaining were acquired sequentially and could be visualized separately or in overlay (Figure 2 ).
For counts of total neuron numbers in LSO, confocal optical sections were acquired with a Leica TCS SP5-2 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) with a Plan Fl20x/0.70 NA objective for the MAP2 stain. Stacks of eight-bit grayscale images were obtained with an axial distance of 3 µm between optical sections each averaged from four successive scans. Finer details (Figure3B ) were taken with a Plan 63x/NA1.32 oil immersion objective. For each optical section the images of one or two fluorochromes were collected sequentially. RGB stacks, montages of RGB optical sections, and maximum-intensity projections were assembled into tables by using ImageJ 1.37k plugins (NIH, USA) and Photoshop (CS6, Adobe Systems, San Jose, CA, USA). Figure images were arranged using CorelDRAW X6 (Corel Corporation, Ottawa, ON, Canada).
For counts of total neuron numbers in LSO, confocal optical sections were acquired with a Leica TCS SP5-2 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) with a Plan Fl20x/0.70 NA objective for the MAP2 stain. Stacks of eight-bit grayscale images were obtained with an axial distance of 3 µm between optical sections each averaged from four successive scans. Finer details (Figure
9
Podoplanin Immunostaining for Lymphatic Mapping
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A 200 section series was cut on a paraffin microtome at a thickness of 5 µm for both lung samples. Every 10th section was immunostained for lymphatic endothelial cells. This was achieved by using D240 (Abcam, Cambridge UK: ab77854) mouse monoclonal anti-human podoplanin primary antibody following a standard immunohistochemistry protocol. To confirm specific staining of the D240 antibody, anti-CD31 (Abcam: ab28364) and anti-pan-keratin (Sigma-Aldrich Company Ltd., Dorset, England: C-2562) were used to stain blood vessels and airways respectively on serial sections (see supplementary material page 1). Negative controls (omitting primary antibody) were included in each staining run.
An Olympus dotslide (Olympus, Southend-on-Sea, UK) system automatically obtained multiple images of the D240 stained slides at 20X magnification and digitised the resulting image as a VSI file. OlyVia software (Olympus) was used to view the slide images.
An Olympus dotslide (Olympus, Southend-on-Sea, UK) system automatically obtained multiple images of the D240 stained slides at 20X magnification and digitised the resulting image as a VSI file. OlyVia software (Olympus) was used to view the slide images.
10
In Situ Hybridization of Bmp2 and Ptgs2
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In situ hybridization was performed using the RNAscope 2.5HD Brown Assay (ACD, 322371) kit, using probes against mouse Bmp2 and Ptgs2, according to manufacturer’s instruction. Bright-field images were captured using DotSlide (Olympus).
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